Abstract
Recent studies have shown telomere-length shortening in colorectal carcinoma. However, to date, no prospective, epidemiological data are available on examining mean leukocyte telomere length as a risk predictor. Using leukocyte DNA samples collected at baseline in a prospective cohort of 14,916 initially healthy American men, we examined the relationship of mean telomere repeat copy number to single gene copy number (T/S ratio), using a modified quantitative polymerase chain reaction protocol, amongst 191 incident colorectal carcinoma cases, matched to 306 controls by age, smoking status, and length of follow-up; all white males. An inverse correlation between T/S ratio and age was observed in our sample population (p=0.038). However, the T/S ratios were similar between cases and controls (p=0.650). Furthermore, in a multivariable adjusted analysis, we found no evidence for an association of the observed T/S ratios with colorectal carcinoma risk (adjusted odds ratio=1.249, 95%CI=0.863-1.808, p=0.238). In summary, the present investigation found no evidence for an association of leukocyte mean telomere length with risk of incident colorectal carcinoma, and further suggests that leukocyte mean telomere length may not be a useful indicator for risk assessment.
Keywords: mean leukocyte telomere length, colorectal carcinoma, risk predictor
Introduction
Telomeres are tandem repeats of DNA sequences —special chromatin structures— located at the ends of eukaryotic chromosomes. These structures are believed to protect the telomeric regions from recombination and degradation, thus avoiding a DNA damage cellular response (1, 2). Genome instability is a hallmark of tumourogenesis, and is a wildly accepted view as a major contribution to the development of cancer, including colorectal carcinoma (CRC) (1, 3). Furthermore, recent studies have implicated telomere length shortening as an independent marker for the progression and/or prognosis of CRC (4-11) based on the comparison of paired cancerous and adjacent non-cancerous tissue specimens from the same individuals. However, to date, no studies have been conducted to examine the relationship of telomere length as a risk predictor with incident CRC.
We thus prospectively examined the possible association of mean peripheral blood leukocyte (PBL) telomere length with risk of incident CRC using a nested, matched case-control sample drawn from the prospective Physicians' Health Study (PHS) cohort.
Materials and Methods
Study Design
We employed a nested case-control design within the PHS, a randomized, double-blinded, placebo-controlled trial of aspirin and beta-carotene initiated in 1982 among 22,071 male, predominantly white (>94%), U.S. physicians, 40 to 84 years of age at study entry (12). Before randomization, 14,916 participants provided an EDTA-anticoagulated blood sample that was stored for genetic analysis. All participants were free of prior myocardial infarction (MI), stroke, transient ischemic attacks, deep venous thrombosis/pulmonary embolism and cancer at study entry. Yearly follow-up self-report questionnaires provided reliable updated information on newly developed diseases. For all reported incident CRC events occurring after study enrollment, medical records were requested and reviewed by an end-points committee.
The nested case-control design has been previously described (13). For each case, one or two controls matched by age±2 years, smoking history (never, past, or current) and length of follow-up were chosen among those subjects who remained free of CRC. The present study consisted of white males only: 76 1-to-1 matched pairs, and 115 1-to-2 matched pairs. Median length of follow-up since randomization for the cases was 6.02 years (interquartile range: 3.22-8.95). The study was approved by the Brigham and Women's Hospital Institutional Review Board for Human Subjects Research.
Mean Telomere Length Determination
Unified Quantitative Polymerase Chain Reaction Assay
Genomic DNA was extracted from whole blood using the QIAmp Spin Column protocol (Qiagen, Chatsworth, CA). Telomere length was determined by a previously described and validated, unified quantitative polymerase chain reaction (qPCR) protocol (14). In brief, two master mixes of PCR reagents were prepared, one for telomere reaction and one for single-copy gene reaction (36B4 on chromosome 12). Telomere repeat copy number to single gene copy number (T/S) ratio was determined on an ABI 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) in a 384-well format using the following PCR protocol: 95°C for 15 minutes to activate Taq-polymerase; 40 cycles of denaturation at 95°C for 15 seconds, and annealing-extension at 54°C for 2 minutes. Each 5uL amplification reaction volume contained 1× Qiagen Quantitect Sybr Green Master Mix (Qiagen, Chatsworth, CA) and 10ng of template DNA. The primer sequences used were described elsewhere (14, 15). All samples for both the telomere and single-copy gene amplifications were done in duplicate on the same 384-well plate. Ct-value assignment was carried out by two independent observers, and if necessary, a complete re-genotyping was performed. The Ct values generated were used to calculate the T/S ratio for each sample using the equation: T/S=2-ΔCt (where ΔCt=Ct single-copy gene-Ct telomere). Results were scored blinded as to case-control status.
Statistical Analysis
As previously noted, the observed telomere-repeat copy number to single gene copy number ratios (T/S ratios) had a skewed distribution, the data were loge-transformation. The T/S ratios between cases and controls were compared using the non-paired t-test. Spearman's correlation analysis was used to assess the effects of age, current smoking, bodymass index (BMI), alcohol use, and exercise on relative T/S ratios amongst all subjects. Risk ratio of CRC associated with loge-transformed T/S ratios were calculated by conditional logistic regression analysis, adjusting for age, smoking status, and length of follow-up since randomization, and further controlling for randomized treatment assignment, BMI, alcohol use (daily/weekly/rarely), and exercise (daily/weekly/rarely). All analyses were carried out using SAS 9.1 package [SAS Institute Inc., Cary, NC]. A two-tailed p-value of 0.05 was considered a statistically significant result.
Results
Baseline characteristics of the study participants are shown in Table 1. In concordance with published data, an inverse relationship of the observed T/S ratios with age amongst all subjects was found (Spearman correlation=−0.093, p=0.038; Supplemental Data Table 1), but not BMI, current smoking, daily alcohol use, nor daily exercise (Supplementary Data Table 1). However, the observed T/S ratios were similar between cases and controls (p=0.650; Table 1). Furthermore, no association of mean T/S ratios with risk of incident CRC was found in the regression analysis (adjusted odds ratio=1.249, 95%CI=0.863-1.808, p=0.238; Table 2). Stratified analysis by median follow-up time since randomization was also performed, and again similar null findings were observed. The coefficients of variation of the telomere, single-gene, and T/S ratio duplicate assays were all <2%, respectively.
Table 1.
Baseline characteristics of study participants.
| Controls (N=306) |
Cases (N=191) |
p** | |
|---|---|---|---|
| Age (years) | 58.05±7.95 | 60.52±8.66 | 0.0012 |
| Smoking Status (%) | 0.67 | ||
| Never | 39.22 | 35.60 | |
| Past | 54.25 | 56.54 | |
| Current | 6.53 | 7.86 | |
| Body-mass Index (kg/m2) | 24.82±2.55 | 25.19±2.86 | 0.14 |
| Alcohol use (%) | 0.59 | ||
| Daily | 29.93 | 31.41 | |
| Weekly | 47.37 | 49.74 | |
| Rarely | 22.70 | 18.85 | |
| Exercise (%) | 0.96 | ||
| Daily | 6.58 | 6.88 | |
| Weekly | 67.44 | 68.26 | |
| Rarely | 25.99 | 24.86 | |
| Aspirin use (%) | 53.27 | 50.26 | 0.51 |
| Beta-carotene use (%) | 50.65 | 53.93 | 0.48 |
| Median length of follow-up since randomization (years)* | 6.60 [3.80-9.19] | 6.02 [3.22-8.95] | 0.30 |
| Cancer site (%) | -- | ||
| Colon | -- | 78.79 | |
| Rectum | -- | 21.21 | |
| Loge-transformed T/S ratio | 3.51±0.58 | 3.53±0.61 | 0.65 |
Mean±SD unless otherwise stated.
T/S=Telomere repeat copy number to single gene copy number.
Median and interquartile range.
Continuous and categorical variables were tested by non-paired t-test and Chi-square analysis, respectively.
Table 2.
Conditional logistic regression analysis of shortening of loge-transformed mean telomere length.
| T/S ratio | Crude | Adjusted |
|---|---|---|
| OR, 95%CI, p | OR, 95%CI, p | |
| Colorectal carcinoma | 0.837, 0.584-1.198, 0.331 | 0.801, 0.553-1.159, 0.238 |
Crude=adjusting for age, smoking status, length of follow-up.
Adjusted=further controlling for randomized treatment group, body-mass index, alcohol use, and exercise.
Discussion
To the best of our knowledge, the present nested, matched case-control investigation is the first to examine the relationship of mean leukocyte telomere length with risk of incident CRC, and we found no evidence for an association. In concordance with previous reports, the present study found an inverse correlation between mean leukocyte telomere length and age.
Recent studies have shown telomere length shortening as an independent marker for the progression and/or prognosis of CRC (4-11). However, these studies were based on telomere length measurements in colonocytes of paired cancerous-noncancerous tissue specimens from the same individuals, as opposed to PBL as used in the current investigation. As previously noted, Craig et al. found no correlation of telomere length with age in normal colonocytes from normal individuals; however, telomere shortening with age was observed in other tissues including blood (16). Furthermore, the study by O'Sullivan et al. also observed an association between telomere length in PBL and gastric tissue, but not between PBL and colon (10). Taken altogether, these data suggest that telomere dynamics in colonocytes differ from other tissues including PBL (10, 16), and may be partly related to the dynamics of telomere-telomerase complex in cell proliferation (8), and exposure/responses to oxidative damage (17). As no prospective, epidemiological data on mean PBL telomere length and risk of incident CRC are available, a cross-reference comparison cannot be achieved on the present null findings in relation to CRC risk. Nevertheless, the present investigation suggests that PBL telomere-length may not play a role in the underlying pathogenesis of CRC.
The nature of the present investigation in which the determination of a case status was based solely on the subsequent development of disease rather than on any arbitrary selection criteria designed by the investigators, greatly reduce the possibility of bias and confounding. Nonetheless, our study population consists of white males only so the data cannot be generalized to other ethnic groups, women, and populations with different socioeconomic status. Furthermore, no baseline information on colorectal screening is available in the present sample population (for example, removal of polyp during colonoscopy). Thus, the potential confounding effect of colorectal screening cannot be assessed in the present context. In our study, we had the ability to detect, based on the present sample size, assuming 80% power, at an alpha of 0.05, a true difference in the mean telomere length ratio of <-0.153 or >0.153. Because of the stringent matching criteria used, 76 cases could only be matched to one control; thus power to detect differences may differ by matched case-control subgroups.
As telomere biology represents a rapidly expanding research field, further thoughts on future development of qPCR technique is worthwhile. Of relevant note, a multiplex monochrome qPCR method for telomere length measurement has recently been described (18). Thus, future methodological comparison to the gold-standard Southern Blot method is worthwhile.
In conclusion, the present prospective, nested case-control study of middle-aged white US men found no evidence for an association of leukocyte mean telomere length with risk of incident colorectal carcinoma. If corroborated in future prospective studies, our present findings further suggest that mean leukocyte telomere length measurement may not be a useful indicator for risk assessment.
Acknowledgments
Supported by grants from the National Institutes of Health (CA-34944, CA-40360, HL-26490, HL-34595).
Footnotes
Conflict-Of-Interest Disclosure: None
References
- 1.Charames GS, Bapat B. Genomic instability and cancer. Curr Mol Med. 2003;3:589–96. doi: 10.2174/1566524033479456. [DOI] [PubMed] [Google Scholar]
- 2.Londono-Vallejo JA. Telomere instability and cancer. Biochimie. 2008;90:73–82. doi: 10.1016/j.biochi.2007.07.009. [DOI] [PubMed] [Google Scholar]
- 3.Bisoffi M, Heaphy CM, Griffith JK. Telomeres: prognostic markers for solid tumors. Int J Cancer. 2006;119:2255–60. doi: 10.1002/ijc.22120. [DOI] [PubMed] [Google Scholar]
- 4.Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, Allshire RC. Telomere reduction in human colorectal carcinoma and with ageing. Nature. 1990;346:866–8. doi: 10.1038/346866a0. [DOI] [PubMed] [Google Scholar]
- 5.Engelhardt M, Albanell J, Drullinsky P, et al. Relative contribution of normal and neoplastic cells determines telomerase activity and telomere length in primary cancers of the prostate, colon, and sarcoma. Clin Cancer Res. 1997;3:1849–57. [PubMed] [Google Scholar]
- 6.Engelhardt M, Drullinsky P, Guillem J, Moore MA. Telomerase and telomere length in the development and progression of premalignant lesions to colorectal cancer. Clin Cancer Res. 1997;3:1931–41. [PubMed] [Google Scholar]
- 7.Tatsumoto N, Hiyama E, Murakami Y, et al. High telomerase activity is an independent prognostic indicator of poor outcome in colorectal cancer. Clin Cancer Res. 2000;6:2696–701. [PubMed] [Google Scholar]
- 8.Gertler R, Rosenberg R, Stricker D, et al. Telomere length and human telomerase reverse transcriptase expression as markers for progression and prognosis of colorectal carcinoma. J Clin Oncol. 2004;22:1807–14. doi: 10.1200/JCO.2004.09.160. [DOI] [PubMed] [Google Scholar]
- 9.Garcia-Aranda C, de Juan C, Diaz-Lopez A, et al. Correlations of telomere length, telomerase activity, and telomeric-repeat binding factor 1 expression in colorectal carcinoma. Cancer. 2006;106:541–51. doi: 10.1002/cncr.21625. [DOI] [PubMed] [Google Scholar]
- 10.O'Sullivan J, Risques RA, Mandelson MT, et al. Telomere length in the colon declines with age: a relation to colorectal cancer? Cancer Epidemiol Biomarkers Prev. 2006;15:573–7. doi: 10.1158/1055-9965.EPI-05-0542. [DOI] [PubMed] [Google Scholar]
- 11.Raynaud CM, Jang SJ, Nuciforo P, et al. Telomere shortening is correlated with the DNA damage response and telomeric protein down-regulation in colorectal preneoplastic lesions. Ann Oncol. 2008;19:1875–81. doi: 10.1093/annonc/mdn405. [DOI] [PubMed] [Google Scholar]
- 12.Physician's health study: aspirin and primary prevention of coronary heart disease. N Engl J Med. 1989;321:1825–8. doi: 10.1056/NEJM198912283212610. [DOI] [PubMed] [Google Scholar]
- 13.Ma J, Stampfer MJ, Giovannucci E, et al. Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer. Cancer Res. 1997;57:1098–102. [PubMed] [Google Scholar]
- 14.Zee RY, Michaud SM, Germer S, Ridker PM. Association of shorter mean telomere length with risk of incident myocardial infarction: A prospective, nested case-control approach. Clinica Chimica Acta. 2009;403:139–41. doi: 10.1016/j.cca.2009.02.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Cawthon RM. Telomere measurement by quantitative PCR. Nucleic Acids Res. 2002;30:e47. doi: 10.1093/nar/30.10.e47. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Craig WL, McKinlay A, Vickers MA. Cellular turnover of normal gastrointestinal epithelium assessed by changes in telomeric: total DNA signal ratios. Eur J Gastroenterol Hepatol. 2003;15:1195–201. doi: 10.1097/00042737-200311000-00008. [DOI] [PubMed] [Google Scholar]
- 17.von Zglinicki T. Role of oxidative stress in telomere length regulation and replicative senescence. Ann N Y Acad Sci. 2000;908:99–110. doi: 10.1111/j.1749-6632.2000.tb06639.x. [DOI] [PubMed] [Google Scholar]
- 18.Cawthon RM. Telomere length measurement by a novel monochrome multiplex quantitative PCR method. Nucleic Acids Res. 2009;37:e21. doi: 10.1093/nar/gkn1027. [DOI] [PMC free article] [PubMed] [Google Scholar]